We are continuing our study of the E. coli bacteriophage T4 model system for duplex DNA replication in which efficient DNA replication in vitro is achieved with purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, the genes 41 and 61 primase-helicase, RNase H, and DNA ligase. We have used DNA primers bearing photoactivated crosslinking residues to label 5 proteins of the T4 DNA replication complex: polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA binding protein. We have determined the relative positions of each of the 5 proteins and the length of DNA in contact with the proteins. We find that the polymerase is bound at the primer terminus and covers 5-7 bases of duplex DNA. Efficient labeling of the polymerase requires all 5 proteins and ATP. When the crosslinkable residue is 14-20 bases from the 31 end of the primer-terminus, the 45 accessory protein is labeled, but only,in the presence of all 5 proteins and ATP. When the primer-terminus is 25 bases from the crosslinkable residue, labeling of the 45 protein is greatly decreased. Our data suggest that the 44/62 complex resides between the polymerase and the 45 proteins. Identification of a T4 Gene Encoding the RNase H Which Removes RNA Primers on the Lagging Strand. We have cloned the T4 DNA encoding an open reading frame which matches the N-terminal sequence of an RNase H which we previously purified from T4 infected cells. Plasmids containing this gene, located upstream of gene 33, express a protein whose size and RNase H and 5' to 3' DNA exonuclease activities are identical to those of the phage-encoded enzyme. This 35 kd protein has three regions with significant amino acid sequence similarity to those of the 5' to 3' exonuclease domain of E. coli pol I and the T7 gene 6 exonuclease. Essential Regions of T4 DNA Polymerase. T4 DNA polymerase has six regions which share sequence similarity with eukaryotic DNA polymerases. To determine the function of shared and unique regions, we are characterizing altered T4 DNA polymerases produced by site-specific mutagenesis of the cloned gene.